CN116047081A

CN116047081A - Kit and method for detecting Abeta 40 and Abeta 42

Info

Publication number: CN116047081A
Application number: CN202310032728.1A
Authority: CN
Inventors: 陈卓; 王咚咚; 王鹏
Original assignee: Inarian Neurodiagnostics LLC
Current assignee: Inarian Neurodiagnostics LLC
Priority date: 2023-01-10
Filing date: 2023-01-10
Publication date: 2023-05-02
Anticipated expiration: 2043-01-10
Also published as: CN116047081B

Abstract

The application discloses a kit for detecting beta-amyloid 40, which comprises: magnetic beads coated with a first antibody targeting beta-amyloid 40; and a chemiluminescent-labeled secondary antibody targeting beta-amyloid 40; wherein the magnetic beads are tosyl activated magnetic beads or carboxyl magnetic beads. Also disclosed is a kit for detecting beta-amyloid 42, a kit for detecting beta-amyloid 40 and beta-amyloid 42, the use of any of the foregoing kits for the preparation of a product for detecting beta-amyloid and/or for diagnosing Alzheimer's disease, a method for detecting the content of beta-amyloid 40 in a sample and a method for detecting the content of beta-amyloid 42 in a sample.

Description

Kit and method for detecting Abeta 40 and Abeta 42

Technical Field

The application belongs to the technical field of immunoassay, and particularly relates to a kit for detecting beta-amyloid 40, a method for detecting beta-amyloid 40, a kit for detecting beta-amyloid 42 and a method for detecting beta-amyloid 42.

Background

Alzheimer's Disease (AD) is one of the most common neurodegenerative diseases associated with age. China is the country with the largest number of AD patients worldwide, and by 2021, china has over 1000 ten thousand AD patients, accounting for about 1/4 of AD patients worldwide. The Chinese Alzheimer's disease cost is 1.47% of the total domestic production value (GDP). Therefore, AD will become one of the most serious diseases threatening the life and property safety and mental health of people in China in the 21 st century.

Alzheimer's Disease (AD) is characterized by neuronal death, often associated with the appearance of amyloid plaques and neurofibrillary tangles (NFT). Among them, amyloid beta (aβ) refers to a group of hydrophobic peptides consisting of 39-43 amino acid residues, mainly aβ42 and aβ40, whose pathological aggregation is associated with neuronal degeneration and cognitive decline in AD. The current research results demonstrate that using the cerebrospinal fluid aβ42/aβ40 ratio as an AD biomarker can effectively increase the percentage of correctly diagnosed patients. Compared with imaging means such as PET-CT, the detection of the biomarker combination can be helpful for early detection of AD and dynamic tracking of therapeutic effects.

In the prior art, the detection of the Abeta 42 and the Abeta 40 mainly adopts the technical platforms of enzyme-linked immunity, POCT, electrochemiluminescence, chemiluminescence and the like.

The prior art for preparing the antibody has the following two main defects:

(1) Insufficient antibody sensitivity

In the prior art, the affinity of the Abeta 42 humanized antibody of the invention such as Jiubao Tian Lifu is only 30-71 nM (BIAcore measurement). The affinity of the anti-N3 pGlu recombinant antibodies of the invention, R.B. Demartos et al, was 0.35nM and 0.71nM (BIAcore assay). Other similar inventive data show that the Abeta antibody sensitivities are all on the order of 0.1-10 nM. Insufficient antibody sensitivity will directly affect the sensitivity of the detection reagent, while reducing the specificity and anti-interference of the detection, and ultimately affecting the reliability of the detection result.

(2) The traditional monoclonal preparation process has congenital deficiency

In the research and development and production process of the in-vitro diagnostic reagent, the repeatability of the products among batches is required to be ensured to be controllable. The difference in the batch-to-batch performance of the antibodies, which are one of the core materials, will be directly reflected in the batch-to-batch reproducibility of the kit. In the preparation process of the murine monoclonal antibody, the antibody titer is easily affected by individual differences of animals. Meanwhile, the volume of the abdominal water of the mice is generally less than 5mL. Although monoclonal antibodies obtained in this manner may have a greater affinity, there are difficulties in preparing large batches of antibodies with stable inter-batch properties by conventional monoclonal antibody preparation processes.

The defects of the existing kit preparation technology mainly comprise the following three points:

(1) The detection time is too long

ELISA platform procedure is relatively tedious, time consuming and laborious. Even if a full-automatic enzyme-linked immunosorbent assay workstation is matched, the incubation time of the sample and the enzyme-labeled antibody cannot be reduced. Current ELISA-based aβ42 and aβ40 detection techniques take generally from 100min to 180min. While similar conditions exist for electrochemical luminescence platforms. Zhou Yanli et al require a 1h incubation time of the sensor with the aβ oligomer and up to 3h incubation time with the AuNPs conjugate. The method of Gu Nengqin also requires at least 2 hours or more for incubation. Such detection duration clearly presents a hurdle for high throughput, high efficiency aβ42 and aβ40 detection in clinical laboratory/physical examination departments.

(2) The detection limit value is high

The lower the detection limit value, the higher the sensitivity of the kit, and the more reliable the quantitative detection of the low-abundance detected object can be achieved. However, luo Haiming et al patent data show that the detection limit of ELISA kits is greater than 100pg/mL. The results of POCT platform, dai Zhengqian and Luo Haiming show that the detection limit of Abeta 42 of the test strip is larger than 40pg/mL, and the sensitivity of the kit is low.

(3) Narrow linear range

The wider linear range ensures that the sample can be detected without dilution, so that the detection times can be reduced and detection deviation caused by sample dilution can be eliminated. Zhang Yuji et al show that the linear range of Aβ42 is only 0-1200pg/mL. Whereas Li Junzun et al establish an Aβ42 detection system with a linear range of only 4.86-1017.68pg/mL. The upper limit of the linear range is lower than the high value of Aβ42 in the article report sample (1456+ -23.01 pg/mL).

Disclosure of Invention

Based on the above description, current aβ42 and aβ40 detection systems have many drawbacks. Early detection of AD and dynamic tracking of therapeutic effects is therefore limited. In order to overcome the defects in the prior art, the application provides a kit for detecting beta-amyloid 40, a kit for detecting beta-amyloid 42, a kit for detecting beta-amyloid 40 and beta-amyloid 42, application of the kit in preparation of products for detecting beta-amyloid and/or for diagnosing Alzheimer's disease, a method for detecting beta-amyloid 40 content in a sample and a method for detecting beta-amyloid 42 content in the sample.

The specific technical scheme of the application is as follows:

1. a kit for detecting β -amyloid 40, comprising:

magnetic beads coated with a first antibody targeting beta-amyloid 40; and

a chemiluminescent-labeled second antibody targeting beta-amyloid 40;

wherein the magnetic beads are tosyl activated magnetic beads or carboxyl magnetic beads.

2. The kit according to claim 1, wherein the mass ratio of the first antibody targeting β -amyloid 40 to the magnetic beads is 1 (20-60), preferably 1 (25-50).

3. The kit according to claim 1 or 2, wherein the ratio of the amount of the second antibody targeting β -amyloid 40 to the amount of the substance of the chemiluminescent agent is 1 (5-15), preferably 1 (7-12).

4. The kit of any one of claims 1 to 3, further comprising a beta-amyloid 40 standard, a substrate, a coating buffer, a wash solution, a blocking solution, and a sample diluent.

5. The kit according to any one of the claims 1 to 4, wherein,

the first antibody targeting beta-amyloid 40 comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3), wherein:

The amino acid sequence of the CDR-H1 is shown as SEQ ID No. 1;

the amino acid sequence of the CDR-H2 is shown as SEQ ID No. 3;

the amino acid sequence of the CDR-H3 is shown as SEQ ID No. 5;

the amino acid sequence of the CDR-L1 is shown as SEQ ID No. 7,

the amino acid sequence of the CDR-L2 is shown as SEQ ID No. 9;

the amino acid sequence of the CDR-L3 is shown as SEQ ID No. 11;

the second antibody targeting beta-amyloid 40 comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3), wherein:

the amino acid sequence of the CDR-H1 is shown as SEQ ID No. 2;

the amino acid sequence of the CDR-H2 is shown as SEQ ID No. 4;

the amino acid sequence of the CDR-H3 is shown as SEQ ID No. 6;

the amino acid sequence of the CDR-L1 is shown as SEQ ID No. 8;

the amino acid sequence of the CDR-L2 is shown as SEQ ID No. 10;

the amino acid sequence of the CDR-L3 is shown as SEQ ID No. 12.

6. The kit according to any one of claims 1 to 5, wherein,

the first antibody targeting beta-amyloid 40 comprises a heavy chain variable region and a light chain variable region, wherein: the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 13, or the amino acid sequence has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% identity with SEQ ID No. 13; the amino acid sequence of the light chain variable region is shown as SEQ ID No. 15, or the amino acid sequence has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID No. 15;

The second antibody targeting beta-amyloid 40 comprises a heavy chain variable region and a light chain variable region, wherein: the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 14, or the amino acid sequence has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% identity with SEQ ID No. 14; the amino acid sequence of the light chain variable region is shown as SEQ ID No. 16, or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID No. 16.

7. The kit according to any one of claims 1 to 6, which is used for diagnosing alzheimer's disease.

8. Use of the kit of any one of items 1 to 7 in the preparation of a product for detecting beta-amyloid 40 and/or for diagnosing alzheimer's disease.

9. A method for detecting the amount of beta-amyloid 40 in a sample comprising the steps of:

mixing a tested sample, magnetic beads coated by a first antibody targeting beta-amyloid 40 and a second antibody labeled by a chemiluminescent agent and targeting beta-amyloid 40, and performing magnetic separation after incubation;

adding a substrate after washing, and measuring the luminescence value of a measured sample;

Fitting a standard curve by using the luminous value of the beta-amyloid 40 standard substance, and substituting the luminous value of the measured sample into an equation to calculate the content of the beta-amyloid 40 in the measured sample;

10. The method according to claim 9, wherein the mass ratio of the first antibody targeting β -amyloid 40 to the magnetic beads is 1 (20-60), preferably 1 (25-50);

preferably, the ratio of the amount of the second antibody targeting beta-amyloid 40 to the amount of the chemiluminescent agent is 1 (5-15), preferably 1 (7-12).

11. The method of claim 9 or 10, wherein the sample to be tested is cerebrospinal fluid;

preferably, the sample to be tested is at least 50. Mu.L, preferably at least 100. Mu.L;

more preferably, 0.005 to 0.015. Mu.g of the primary antibody targeting beta-amyloid 40 and 0.15 to 0.25ng of the secondary antibody targeting beta-amyloid 40 are required per microliter of test sample;

further preferably, the incubation time is at least 15min.

12. The method according to any one of claims 9 to 11, wherein the first antibody targeting β -amyloid 40 and the second antibody targeting β -amyloid 40 labeled with a chemiluminescent agent are the first antibody targeting β -amyloid 40 and the second antibody targeting β -amyloid 40 labeled with a chemiluminescent agent described in item 5 or 6.

13. A kit for detecting β -amyloid 42, comprising:

magnetic beads coated with a first antibody targeting beta-amyloid 42; and

a chemiluminescent-labeled second antibody targeting beta-amyloid 42;

14. The kit according to claim 13, wherein the mass ratio of the first antibody targeting β -amyloid 42 to the magnetic beads is 1 (20-60), preferably 1 (25-50).

15. The kit according to claim 13 or 14, wherein the ratio of the amount of the second antibody targeting β -amyloid 42 to the amount of the substance of the chemiluminescent agent is 1 (5-15), preferably 1 (7-12).

16. The kit of any one of claims 13 to 15, further comprising a beta-amyloid 42 standard, a substrate, a coating buffer, a wash solution, a blocking solution, and a sample diluent.

17. The kit according to any one of claims 13 to 16, wherein,

the first antibody targeting beta-amyloid 42 comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3), wherein:

The amino acid sequence of the CDR-H1 is shown as SEQ ID No. 17;

the amino acid sequence of the CDR-H2 is shown as SEQ ID No. 19;

the amino acid sequence of the CDR-H3 is shown as SEQ ID No. 21;

the amino acid sequence of the CDR-L1 is shown as SEQ ID No. 23,

the amino acid sequence of the CDR-L2 is shown as SEQ ID No. 25;

the amino acid sequence of the CDR-L3 is shown as SEQ ID No. 27;

the second antibody targeting beta-amyloid 42 comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3), wherein:

the amino acid sequence of the CDR-H1 is shown as SEQ ID No. 18;

the amino acid sequence of the CDR-H2 is shown as SEQ ID No. 20;

the amino acid sequence of the CDR-H3 is shown as SEQ ID No. 22;

the amino acid sequence of the CDR-L1 is shown as SEQ ID No. 24;

the amino acid sequence of the CDR-L2 is shown as SEQ ID No. 26;

the amino acid sequence of the CDR-L3 is shown as SEQ ID No. 28.

18. The kit according to any one of claims 13 to 17, wherein,

the first antibody targeting beta-amyloid 42 comprises a heavy chain variable region and a light chain variable region, wherein: the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 29, or the amino acid sequence has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% identity with SEQ ID No. 29; the amino acid sequence of the light chain variable region is shown as SEQ ID No. 31 or an amino acid sequence which has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% identity with SEQ ID No. 31;

The second antibody targeting beta-amyloid 42 comprises a heavy chain variable region and a light chain variable region, wherein: the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 30, or the amino acid sequence has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% identity with SEQ ID No. 30; the amino acid sequence of the light chain variable region is shown as SEQ ID No. 32, or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID No. 32.

19. The kit according to any one of claims 13 to 18, which is used for diagnosing alzheimer's disease.

20. Use of a kit according to any one of claims 13 to 19 for the preparation of a product for the detection of beta-amyloid 42 and/or for the diagnosis of alzheimer's disease.

21. A method for detecting the amount of beta-amyloid 42 in a sample comprising the steps of:

mixing a tested sample, magnetic beads coated by a first antibody targeting beta-amyloid 42 and a second antibody targeting beta-amyloid 42 marked by a chemiluminescent agent, and performing magnetic separation after incubation;

Fitting a standard curve by using the luminous value of the beta-amyloid 42 standard substance, and substituting the luminous value of the measured sample into an equation to calculate the content of the beta-amyloid 42 in the measured sample;

22. The method according to claim 21, wherein the mass ratio of the first antibody targeting β -amyloid 42 to the magnetic beads is 1 (20-60), preferably 1 (25-50);

preferably, the ratio of the amount of the second antibody targeting beta-amyloid 42 to the amount of the chemiluminescent agent is 1 (5-15), preferably 1 (7-12).

23. The method of claim 21 or 22, wherein the sample to be tested is cerebrospinal fluid;

more preferably, 0.005 to 0.015. Mu.g of the primary antibody targeting beta-amyloid 42 and 0.15 to 0.25ng of the secondary antibody targeting beta-amyloid 42 are required per microliter of test sample;

further preferably, the incubation time is at least 15min.

24. The method of any one of claims 21 to 23, wherein the first antibody targeting β -amyloid 42, the second antibody targeting β -amyloid 42 labeled with a chemiluminescent agent, is the first antibody targeting β -amyloid 42, the second antibody targeting β -amyloid 42 labeled with a chemiluminescent agent, as described in item 17 or 18.

25. A kit for detecting β -amyloid 40 and β -amyloid 42, comprising:

magnetic beads coated with a first antibody targeting beta-amyloid 40;

a chemiluminescent-labeled second antibody targeting beta-amyloid 40;

magnetic beads coated with a first antibody targeting beta-amyloid 42; and

a chemiluminescent-labeled second antibody targeting beta-amyloid 42;

26. The kit of item 25, wherein,

the mass ratio of the first antibody targeting the beta-amyloid 40 to the magnetic beads is 1 (20-60), preferably 1 (25-50);

the mass ratio of the first antibody targeting the beta-amyloid 42 to the magnetic beads is 1 (20-60), preferably 1 (25-50).

27. The kit according to item 25 or 26, characterized in that,

the ratio of the amount of the second antibody targeting the beta-amyloid 40 to the amount of the substance of the chemiluminescent agent is 1 (5-15), preferably 1 (7-12);

the ratio of the amount of the second antibody targeting the beta-amyloid 42 to the amount of the substance of the chemiluminescent agent is 1 (5-15), preferably 1 (7-12).

28. The kit of any one of claims 25 to 27, further comprising a beta-amyloid 40 standard, a beta-amyloid 42 standard, a substrate, a coating buffer, a wash solution, a blocking solution, and a sample diluent.

29. The kit according to any one of claims 25 to 28, wherein,

the first antibody targeting beta-amyloid 40, the second antibody targeting beta-amyloid 40 is the first antibody targeting beta-amyloid 40, the second antibody targeting beta-amyloid 40 described in item 5 or 6;

the first antibody targeting beta-amyloid 42 and the second antibody targeting beta-amyloid 42 are the first antibody targeting beta-amyloid 42 and the second antibody targeting beta-amyloid 42 described in item 17 or 18.

30. The kit according to any one of claims 25 to 29, for use in diagnosing alzheimer's disease.

31. Use of the kit of any one of claims 25-30 in the manufacture of a product for detecting beta-amyloid 40, beta-amyloid 42 and/or for diagnosing alzheimer's disease.

ADVANTAGEOUS EFFECTS OF INVENTION

The kit for detecting the beta-amyloid 40 and/or the beta-amyloid 42 takes a pair of recombinant antibodies as core raw materials, comprises magnetic beads coated with a first antibody for targeting the beta-amyloid and a second antibody for targeting the beta-amyloid, which are labeled by chemiluminescence. The recombinant antibody contained in the kit has high affinity, high sensitivity and obviously shortened production period, and is more suitable for being used as a core raw material in the field of in-vitro diagnostic reagents. The magnetic beads used in the kit and the detection method are specific tosyl activated magnetic beads or carboxyl magnetic beads, the detection limit value is lower, the linear range is wider, and the detection sensitivity is high. In addition, the magnetic particle chemiluminescence platform has the advantages of simplicity in operation, high automation degree, homogeneous reaction system and the like, and the detection time can be further shortened by means of the reaction system of the one-step method. The application further adopts the recombinant antibody with high affinity and stable performance, which has stronger recognition capability to the epitope of the antigen and higher binding degree with the antigen, so that the high-affinity antibody has stronger capturing capability to the low-abundance target antigen, provides a lower detection limit for the kit, and has high detection sensitivity; on the premise of not changing the coating process, the high-affinity antibody can be replaced to enhance the antigen grabbing capacity of unit mass magnetic beads or other mediums, and further the linear range of the reagent is obviously improved on the premise of not generating a Hook effect.

Drawings

FIG. 1 shows A.beta.40 kit and method using the present application

And consistency analysis results of the content of Abeta 40 in the G1200 platform product detection sample.

FIG. 2 shows A.beta.42 kit and method for using the same

And consistency analysis results of the content of Abeta 42 in the G1200 platform product detection sample.

Detailed Description

The following examples are set forth in order to provide a more thorough understanding of the present invention and to fully convey the scope of the present application to those skilled in the art.

Technical and scientific terms used in the present specification have the same meaning as commonly understood by one of ordinary skill in the art, and if so conflict, the present specification will control.

As used herein, the term "recombinant antibody" is an antibody obtained by cloning immunospecific heavy and light chain antibody genes into highly efficient expression vectors, and introducing these vectors into an expression host (e.g., bacteria, yeast or mammal) for antibody expression. The antibodies of the present application may be fully humanized antibodies, chimeric antibodies, and the like.

In some embodiments, the antibodies of the present application are full length antibodies, which typically refer to antibodies consisting of two "heavy chains" and two "light chains. A "heavy chain" is generally a polypeptide consisting of, in the N-to C-terminal direction, a heavy chain variable region (VH), a heavy chain constant region 1 (CH 1), a Hinge Region (HR), an antibody heavy chain constant region 2 (CH 2), and a heavy chain constant region 3 (CH 3), abbreviated as VH-CH1-HR-CH2-CH3; in some embodiments, a "full length antibody heavy chain" is a polypeptide consisting of VH, CH1, HR, CH2, and CH3 in the N-to C-terminal direction. A "full length antibody light chain" is generally a polypeptide consisting of a light chain variable region (VL) in the N-to C-terminal direction, and a light chain constant region (CL), abbreviated VL-CL.

In the present application, the light chain constant region and the heavy chain constant region may be any light chain constant region and heavy chain constant region. The light chain constant region (CL) may be kappa (kappa) or lambda (lambda) and the heavy chain constant region may be any one of the heavy chain constant regions of IgG, igM, igA, igE, igD.

It is well known to those skilled in the art that complementarity determining regions (CDRs, typically CDR1, CDR2 and CDR 3) are regions of the variable region that have the greatest influence on the affinity and specificity of an antibody. In some embodiments, from N-terminal to C-terminal, the heavy chain variable region and the light chain variable region each comprise FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDR sequences of the heavy chain variable region or the light chain variable region are defined in two common ways, namely Kabat definition and Chothia definition, see for example Kabat et al, "Sequences of Proteins ofImmunological Interest", national Institutes ofHealth, bethesda, MD. (1991); al-Lazikani et Al, J Mol Biol 273:927-948 (1997); and Martin et al, proc.Natl.Acad.Sci.USA 86:9268-9272 (1989). For a given antibody variable region sequence, the CDR sequences in the heavy chain variable region or light chain variable region sequence can be determined according to the Kabat definition or Chothia definition. In embodiments of the present application, the CDR sequences are determined using the Chothia definition.

The term "identity" is defined herein as the percentage of identical residues in an amino acid or nucleotide sequence variant after sequence alignment and introduction of gaps, if desired, to achieve a maximum percentage of homology. Methods and computer programs for alignment are well known in the art. In this context, reference to an amino acid sequence having a percent identity to the amino acid sequence refers to a sequence having said percent identity over the entire length of the amino acid sequence referred to.

In this context, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells. The vector may include a vector mainly used for inserting DNA or RNA into a cell, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of transcription and/or translation of DNA or RNA. The carrier also includes a carrier having a plurality of functions as described above. The vector may be a polynucleotide capable of transcription and translation into a polypeptide when introduced into a suitable host cell. Typically, the vector will produce the desired expression product by culturing a suitable host cell comprising the vector.

In this context, the term "nucleic acid" or "polynucleotide" or "nucleic acid molecule" generally refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term may include nucleic acids comprising analogs of natural nucleotides that have similar binding properties as the reference nucleic acid (e.g., sequence information is shown) and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, the sequence of a nucleic acid may include variants thereof that are conservatively modified, such as degenerate codon substitutions, alleles, orthologs, SNPs, and complementary sequences, as well as the sequences explicitly indicated.

As used herein, the term "EC50 value" refers to half maximal effect concentration (concentration for 50%ofmaximal effect,EC50), meaning that concentration that causes 50% maximal effect.

Herein, "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to an inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be determined by the equilibrium dissociation constant (K _D ) And (3) representing. Affinity can be determined by common methods known in the art.

Herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homologous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during production of monoclonal antibody preparations), which are typically present in minor amounts. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being derived from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

Herein, the term "recombinant antibody targeting β -amyloid 40" means a recombinant antibody capable of binding β -amyloid 40 with sufficient affinity, capable of being used as a diagnostic and/or therapeutic agent targeting β -amyloid 40. By "recombinant antibody targeting beta-amyloid 42" is meant a recombinant antibody capable of binding beta-amyloid 42 with sufficient affinity, capable of being used as a diagnostic and/or therapeutic agent for targeting beta-amyloid 42.

The recombinant antibodies targeting beta-amyloid 40 of the present application do not bind to target-independent proteins. Herein, "unrelated proteins" refer to proteins other than the target β -amyloid 40; here, "not bonded" means: where the binding capacity of the recombinant antibody targeting β -amyloid 40 of the present application to β -amyloid 40 as its target is taken as 100%, the binding capacity of the recombinant antibody targeting β -amyloid 40 of the present application to the unrelated protein is less than 10%, e.g. 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.

The recombinant antibodies targeting beta-amyloid 42 of the present application do not bind to target-independent proteins. Herein, "unrelated proteins" refer to proteins other than the target β -amyloid 42; here, "not bonded" means: where the binding capacity of the recombinant antibodies targeting β -amyloid 42 of the present application to β -amyloid 42 as its target is taken as 100%, the binding capacity of the recombinant antibodies targeting β -amyloid 42 of the present application to the unrelated protein is less than 10%, e.g. 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.

Beta-amyloid suitable for antibody production may be produced by any of a variety of standard protein purification or recombinant expression techniques known in the art. Forms of beta-amyloid suitable for generating an immune response include the isoforms Abeta 40 and Abeta 42 of beta-amyloid. Additional forms of beta-amyloid include beta-amyloid expressing cells, beta-amyloid containing preparations or cell extracts or fractions, and partially purified beta-amyloid.

In some embodiments, the present application uses magnetic particle chemiluminescent immunoassay to determine the levels of aβ40, aβ42 in a sample based on the following specific principles: directly labeling antigen or antibody (chemiluminescent agent marker) with chemiluminescent agent, reacting with corresponding antibody or antigen in the sample to be tested, and antigen or antibody with magnetic particle property (magnetic bead), separating the chemiluminescent agent marker in the combined state (precipitation part) and the free state by magnetic field, adding luminescent promoter (luminescent substrate) for luminescent reaction, and quantitatively or qualitatively detecting the luminescent intensity.

In this context, ELISA (enzyme linked immune sorbent assay), i.e. enzyme-linked immunosorbent assay, refers to a detection method that uses the characteristic that an antibody molecule can specifically bind to an antigen molecule to bind a free hetero-protein to a target protein bound to a solid support, and uses a specific label to perform qualitative or quantitative analysis. The principle is that the antigen or antibody can be physically adsorbed on the surface of the solid carrier and maintain the immunological activity; the antigen or antibody is capable of forming an enzyme conjugate with the enzyme via a covalent bond while maintaining the respective immunological or enzymatic activity; after binding the enzyme conjugate to the corresponding antigen or antibody, the occurrence of an immune response can be determined by a color reaction of the added substrate, the color reaction being in direct proportion to the amount of the corresponding antigen or antibody in the sample.

In a first aspect, the present application provides a kit for detecting β -amyloid 40, the kit comprising: magnetic beads coated with a first antibody targeting beta-amyloid 40; and a chemiluminescent-labeled secondary antibody targeting beta-amyloid 40; wherein the magnetic beads are tosyl activated magnetic beads or carboxyl magnetic beads.

Compared with the traditional magnetic beads, the magnetic beads with the superparamagnetic preactivation function have the characteristics of higher magnetic responsiveness, good dispersibility, extremely low nonspecific adsorption, preactivation reaction sites and the like, can be directly used for carrying out high-load combination with various biological ligands (proteins, polypeptides, oligonucleotides, drug molecules and the like), and realizes the purpose of covalent bond coupling on the surfaces of the magnetic beads. The magnetic bead has long hydrophilic interval arm and mild reaction condition, so that it is especially suitable for immobilization of bioactive macromolecule.

The carboxyl magnetic beads have the characteristics of superparamagnetism, quick magnetic responsiveness, rich carboxyl functional groups, monodispersity, submicron-scale particle size and the like, and can be used for covalently coupling biological ligands such as polypeptide, protein, antibody, oligonucleotide and the like to the surfaces of microspheres under the action of special chemical reagents (such as EDC).

In some embodiments, the mass ratio of the first antibody targeting β -amyloid 40 to the magnetic beads is 1 (20-60), e.g., 1:20, 1:22, 1:25, 1:28, 1:30, 1:32, 1:35, 1:37, 1:40, 1:43, 1:45, 1:48, 1:50, 1:52, 1:55, 1:57, 1:60, etc., preferably 1 (25-50).

In some embodiments, the amount of the second antibody that targets β -amyloid 40 to the chemiluminescent agent is 1 (5-15), e.g., can be 5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:10.5, 1:11, 1:11.5, 1:12, 1:12.5, 1:13, 1:13.5, 1:14, 1:14.5, 1:15, etc., preferably 1 (7-12).

In some embodiments, the kit further comprises a beta-amyloid 40 standard, a substrate, a coating buffer, a wash solution, a blocking solution, and a sample diluent, wherein the coating buffer provides a high pH environment for magnetic bead coating, enhancing coating efficiency; the blocking liquid is used for blocking uncoupling empty space and preventing later non-specific binding. In some embodiments, the substrate comprises luminescence excitation solution a and luminescence excitation solution B, wherein luminescence excitation solution a is nitric acid and hydrogen peroxide aqueous solution, luminescence excitation solution B is sodium hydroxide aqueous solution, the coating buffer is borate buffer, and the wash solution is PBS and tween-20; the blocking solution was BSA blocking solution and the sample dilution was PBS buffer. In some embodiments, the coating buffer is 0.1M borate buffer (pH 9.5), the blocking solution is 10% BSA blocking solution, the sample diluent is PBS buffer at pH 7.4, and the sample diluent further comprises 1% BSA and 0.1% Proclin-300 per liter of sample diluent.

In the above embodiment, the kit further comprises a preservation buffer and a quenching buffer, wherein the preservation buffer is used for providing a stable pH environment, weakening antibody degradation, preserving, and the like; the quenching buffer is used to quench off the label that is not bound to the antibody. In some embodiments, the preservation buffer is buffer TBS-T and the quenching buffer is 5% DL-lysine.

In some embodiments, the first antibody targeting β -amyloid 40 comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3), wherein: the amino acid sequence of the CDR-H1 is shown as SEQ ID No. 1 (GFNFKDF), the amino acid sequence of the CDR-H2 is shown as SEQ ID No. 3 (DPENGD), the amino acid sequence of the CDR-H3 is shown as SEQ ID No. 5 (NPQSERKPSTYSL), the amino acid sequence of the CDR-L1 is shown as SEQ ID No. 7 (KSSQSLLYGDGKTYLN), the amino acid sequence of the CDR-L2 is shown as SEQ ID No. 9 (LVSKLDS), and the amino acid sequence of the CDR-L3 is shown as SEQ ID No. 11 (VQGSYFPLT); the second antibody targeting beta-amyloid 40 comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3), wherein: the amino acid sequence of the CDR-H1 is shown as SEQ ID No. 2 (GPNFKDF), the amino acid sequence of the CDR-H2 is shown as SEQ ID No. 4 (DPENGD), the amino acid sequence of the CDR-H3 is shown as SEQ ID No. 6 (CDAIQRHFIDYDF), the amino acid sequence of the CDR-L1 is shown as SEQ ID No. 8 (KSSQSLLYSDGKTYLN), the amino acid sequence of the CDR-L2 is shown as SEQ ID No. 10 (LVSKLDS), and the amino acid sequence of the CDR-L3 is shown as SEQ ID No. 12 (VQESHFPLT).

In some embodiments, the first antibody that targets β -amyloid 40 comprises a heavy chain variable region and a light chain variable region, wherein: the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 13 (EVQ LQQSAAELVRTGASVKLSCTTSGFNFKDFYMHWIKQRPEQGLEWIGWLD PENGDTEYAPKFQGKATMAADTSSDTTYLQLSSLMSEDTAVYYCNVNPQ SERKPSTYSLWGQGTTLTVAS), or an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID No. 13, the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 15 (DVMMTGSPHTLTVTIGQPASISCKSSQSLLYGD GKTYLNWLFQRPGQSPKRLIYLVSKLDSGVPDRFTGSGTGTDFTLKISRVE AEDLGIYYCVQGSYFPLTFGAGTKIALKRA), or an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID No. 15; the second antibody targeting beta-amyloid 40 comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region has the amino acid sequence shown in SEQ ID No. 14 (EVQLQMSAAELVRTGAS VKLSCTTSGPNFKDFYMHWIKQRPEQGLDWIGWLDPENGDTEYAPKFQG KATMAGDTSSDTTYLQLSSLMSEDTAVRYCNVCDAIQRHFIDYDFWGQG TTLTVAS), or has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID No. 14, or has the amino acid sequence shown in SEQ ID No. 16 (DVMMTQSPRTISVTIGQPASISCKSSQSLLYSDGKTYLNWLLQRPG QSPKRLIYLVSKLDSGVPDRFTGSGSGTDPTLKISRVEAEDLGIYYCVQES HFPLTFGAGTWLALKRA), or has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID No. 16.

In some embodiments, the kit for detecting β -amyloid 40 of any one of the preceding claims is used to diagnose alzheimer's disease.

In a second aspect, the present application provides the use of a kit for detecting β -amyloid 40 according to any one of the preceding claims for the preparation of a product for detecting β -amyloid 40 and/or for diagnosing alzheimer's disease.

In a third aspect, the present application also provides a method of detecting the content of β -amyloid 40 in a sample comprising the steps of: mixing a tested sample, magnetic beads coated by a first antibody targeting beta-amyloid 40 and a second antibody labeled by a chemiluminescent agent and targeting beta-amyloid 40, and performing magnetic separation after incubation; adding a substrate after washing, and measuring the luminescence value of a measured sample; fitting a standard curve by using the luminous value of the beta-amyloid 40 standard substance, and substituting the luminous value of the measured sample into an equation to calculate the content of the beta-amyloid 40 in the measured sample; wherein the magnetic beads are tosyl activated magnetic beads or carboxyl magnetic beads.

In some embodiments, the sample to be tested is cerebrospinal fluid, and the sample to be tested may be at least 50. Mu.L, for example, 50. Mu.L, 60. Mu.L, 70. Mu.L, 80. Mu.L, 90. Mu.L, 100. Mu.L, 110. Mu.L, 120. Mu.L, 130. Mu.L, 140. Mu.L, 150. Mu.L, 160. Mu.L, 170. Mu.L, 180. Mu.L, etc., and preferably at least 100. Mu.L, and the incubation time may be at least 15min, for example, 15min, 16min, 17min, 18min, 19min, 20min, etc.

In some embodiments, the test sample is cerebrospinal fluid, the test sample is at least 50. Mu.L, preferably at least 100. Mu.L, and the incubation time is at least 15min, and 0.005-0.015. Mu.g of the first antibody targeting beta-amyloid 40 and 0.15-0.25 ng of the second antibody targeting beta-amyloid 40 are required per microliter of the test sample, and the first antibody targeting beta-amyloid 40 required per microliter of the test sample can be, for example, 0.005. Mu.g, 0.006. Mu.g, 0.007. Mu.g, 0.008. Mu.g, 0.009. Mu.g, 0.01. Mu.g, 0.011. Mu.g, 0.012. Mu.g, 0.013. Mu.g, 0.014. Mu.g, 0.015. Mu.g, and the second antibody targeting beta-amyloid 40 required per microliter of the test sample can be, for example, 0.15ng, 0.16ng, 0.17ng, 0.18ng, 0.19ng, 0.21ng, 0.25ng, and the like.

In some embodiments, the first antibody that targets β -amyloid 40, the second antibody that targets β -amyloid 40 labeled with a chemiluminescent agent, is the first antibody that targets β -amyloid 40 described in the first aspect above, the second antibody that targets β -amyloid 40 labeled with a chemiluminescent agent.

In a fourth aspect, the present application provides a kit for detecting β -amyloid 42, the kit comprising: magnetic beads coated with a first antibody targeting beta-amyloid 42; and a chemiluminescent-labeled secondary antibody targeting beta-amyloid 42; wherein the magnetic beads are tosyl activated magnetic beads or carboxyl magnetic beads.

In some embodiments, the mass ratio of the first antibody targeting β -amyloid 42 to the magnetic beads is 1 (20-60), e.g., 1:20, 1:22, 1:25, 1:28, 1:30, 1:32, 1:35, 1:37, 1:40, 1:43, 1:45, 1:48, 1:50, 1:52, 1:55, 1:57, 1:60, etc., preferably 1 (25-50).

In some embodiments, the amount of the second antibody targeting β -amyloid 42 to the chemiluminescent agent is 1 (5-15), e.g., can be 5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:10.5, 1:11, 1:11.5, 1:12, 1:12.5, 1:13, 1:13.5, 1:14, 1:14.5, 1:15, etc., preferably 1 (7-12).

In some embodiments, the kit further comprises a beta-amyloid 42 standard, a substrate, a coating buffer, a wash solution, a blocking solution, and a sample diluent, wherein the coating buffer provides a high pH environment for magnetic bead coating, enhancing coating efficiency; the blocking liquid is used for blocking uncoupling empty space and preventing later non-specific binding. In some embodiments, the substrate comprises luminescence excitation solution a and luminescence excitation solution B, wherein luminescence excitation solution a is nitric acid and hydrogen peroxide aqueous solution, luminescence excitation solution B is sodium hydroxide aqueous solution, the coating buffer is borate buffer, and the wash solution is PBS and tween-20; the blocking solution was BSA blocking solution and the sample dilution was PBS buffer. In some embodiments, the coating buffer is 0.1M borate buffer (pH 9.5), the blocking solution is 10% BSA blocking solution, the sample diluent is PBS buffer at pH 7.4, and the sample diluent further comprises 1% BSA and 0.1% Proclin-300 per liter of sample diluent.

In some embodiments, the first antibody targeting β -amyloid 42 comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3), wherein: the amino acid sequence of the CDR-H1 is shown as SEQ ID No. 17 (GFNFKDF), the amino acid sequence of the CDR-H2 is shown as SEQ ID No. 19 (DPENGD), the amino acid sequence of the CDR-H3 is shown as SEQ ID No. 21 (HAIWYDEEELYRH), the amino acid sequence of the CDR-L1 is shown as SEQ ID No. 23 (KSSQSLLYSDGKGYLN), the amino acid sequence of the CDR-L2 is shown as SEQ ID No. 25 (LVSKLDS), and the amino acid sequence of the CDR-L3 is shown as SEQ ID No. 27 (VQGSHFPLT); the second antibody targeting beta-amyloid 42 comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3), wherein: the amino acid sequence of the CDR-H1 is shown as SEQ ID No. 18 (GFNFKDF), the amino acid sequence of the CDR-H2 is shown as SEQ ID No. 20 (EPENGD), the amino acid sequence of the CDR-H3 is shown as SEQ ID No. 22 (SENISYLRRTRGA), the amino acid sequence of the CDR-L1 is shown as SEQ ID No. 24 (KSSQTLLYSDGKTYLN), the amino acid sequence of the CDR-L2 is shown as SEQ ID No. 26 (LVSKLDS), and the amino acid sequence of the CDR-L3 is shown as SEQ ID No. 28 (VQGSHFPLT).

In some embodiments, the first antibody targeting β -amyloid 42 comprises a heavy chain variable region and a light chain variable region, wherein: the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 29 (EVQ LQQSAGELVRTGASVKLSCATSGFNFKDFYWHWIKQRPEQGLDWIGWLD PENGDTEYAPKFQGKATMAADTSSDTTYLQLSSLMSEETAVYYCNVHAI WYDEEELYRHWGQGTTLTIAS), or an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID No. 29, the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 31 (DVMWTQSPRTLTVTIGQPASISCKSSQS LLYSDGKGYLNWLFQRPGQSPKRLIYLVSKLDSGVPDRFTGSYSGSDFTL KISRVEAEDLGIYYCVQGSHFPLTFGAGTKLAVKRA), or an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID No. 31; the second antibody targeting beta-amyloid 42 comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region has the amino acid sequence shown in SEQ ID No. 30 (EVQLQTSAGELV RTGASIKLSCATSGFNFKDFYWHWLKQRPEQGLDWIGWLEPENGDTEHA PKFQGKATMAAETSSDTTYLQLSFLMSEETAVYHCNVSENISYLRRTRGA WGQGTTLTIAS), or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID No. 30, or the light chain variable region has the amino acid sequence shown in SEQ ID No. 32 (DVMWTQSPRTLSVTIGQPASISCKSSQSLLYSDGKTYLNWL LQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSNTDFTLKISRVDAEELGIYY CVQGSHFPLTFGAGTKLALKHAD), or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID No. 32.

In some embodiments, the kit for detecting β -amyloid 42 of any one of the preceding claims is used to diagnose alzheimer's disease.

In a fifth aspect, the present application provides the use of a kit for detecting β -amyloid 42 according to any one of the preceding claims for the preparation of a product for detecting β -amyloid 42 and/or for diagnosing alzheimer's disease.

In a sixth aspect, the present application also provides a method of detecting the content of β -amyloid 42 in a sample comprising the steps of: mixing a tested sample, magnetic beads coated by a first antibody targeting beta-amyloid protein 42 and a second antibody labeled by a chemiluminescent agent and targeting beta-amyloid protein 42, and performing magnetic separation after incubation; adding a substrate after washing, and measuring the luminescence value of a measured sample; fitting a standard curve by using the luminous value of the beta-amyloid 42 standard substance, and substituting the luminous value of the measured sample into an equation to calculate the content of the beta-amyloid 42 in the measured sample; wherein the magnetic beads are tosyl activated magnetic beads or carboxyl magnetic beads.

In some embodiments, the test sample is cerebrospinal fluid, the test sample is at least 50. Mu.L, preferably at least 100. Mu.L, and the incubation time is at least 15min, and 0.005-0.015. Mu.g of the first antibody targeting beta-amyloid 42 and 0.15-0.25 ng of the second antibody targeting beta-amyloid 42 are required per microliter of the test sample, and the first antibody targeting beta-amyloid 42 is required to be used per microliter of the test sample, for example, 0.005. Mu.g, 0.006. Mu.g, 0.007. Mu.g, 0.008. Mu.g, 0.009. Mu.g, 0.01. Mu.g, 0.011. Mu.g, 0.012. Mu.g, 0.013. Mu.g, 0.014. Mu.g, 0.015. Mu.g, and the second antibody targeting beta-amyloid 42 is required to be used per microliter of the test sample, for example, 0.15ng, 0.16ng, 0.17ng, 0.18ng, 0.19ng, 0.21ng, 0.25ng, etc.

In some embodiments, the first antibody that targets β -amyloid 42, the second antibody that targets β -amyloid 42 labeled with a chemiluminescent agent, is the first antibody that targets β -amyloid 42 described in the fourth aspect above, the second antibody that targets β -amyloid 42 labeled with a chemiluminescent agent.

In a seventh aspect, the present application provides a kit for detecting β -amyloid 40 and β -amyloid 42, comprising: magnetic beads coated with a first antibody targeting beta-amyloid 40; a chemiluminescent-labeled second antibody targeting beta-amyloid 40; magnetic beads coated with a first antibody targeting beta-amyloid 42; and a chemiluminescent-labeled secondary antibody targeting beta-amyloid 42; wherein the magnetic beads are tosyl activated magnetic beads or carboxyl magnetic beads.

In some embodiments, the mass ratio of the first antibody targeting β -amyloid 40 to the magnetic beads is 1 (20-60), e.g., can be 1:20, 1:22, 1:25, 1:28, 1:30, 1:32, 1:35, 1:37, 1:40, 1:43, 1:45, 1:48, 1:50, 1:52, 1:55, 1:57, 1:60, etc., preferably 1 (25-50); the mass ratio of the first antibody targeting the beta-amyloid 42 to the magnetic beads is 1 (20-60), for example, 1:20, 1:22, 1:25, 1:28, 1:30, 1:32, 1:35, 1:37, 1:40, 1:43, 1:45, 1:48, 1:50, 1:52, 1:55, 1:57, 1:60, etc., preferably 1 (25-50).

In some embodiments, the amount of the second antibody that targets β -amyloid 40 to the substance of the chemiluminescent agent is 1 (5-15), e.g., can be 5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:10.5, 1:11, 1:11.5, 1:12, 1:12.5, 1:13, 1:13.5, 1:14, 1:14.5, 1:15, etc., preferably 1 (7-12); the ratio of the amount of the second antibody targeting the β -amyloid 42 to the amount of the chemiluminescent agent is, for example, 1 (5-15), and may be, for example, 5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:10.5, 1:11, 1:11.5, 1:12, 1:12.5, 1:13, 1:13.5, 1:14, 1:14.5, 1:15, etc., and preferably 1 (7-12).

In some embodiments, the kit further comprises a beta-amyloid 40 standard, a beta-amyloid 42 standard, a substrate, a coating buffer, a wash solution, a blocking solution, and a sample diluent, wherein the coating buffer provides a high pH environment for magnetic bead coating, enhancing coating efficiency; the blocking liquid is used for blocking uncoupling empty space and preventing later non-specific binding. In some embodiments, the substrate comprises luminescence excitation solution a and luminescence excitation solution B, wherein luminescence excitation solution a is nitric acid and hydrogen peroxide aqueous solution, luminescence excitation solution B is sodium hydroxide aqueous solution, the coating buffer is borate buffer, and the wash solution is PBS and tween-20; the blocking solution was BSA blocking solution and the sample dilution was PBS buffer. In some embodiments, the coating buffer is 0.1M borate buffer (pH 9.5), the blocking solution is 10% BSA blocking solution, the sample diluent is PBS buffer at pH 7.4, and the sample diluent further comprises 1% BSA and 0.1% Proclin-300 per liter of sample diluent.

In some embodiments, the first antibody targeting β -amyloid 40 comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3), wherein: the amino acid sequence of the CDR-H1 is shown as SEQ ID No. 1 (GFNFKDF), the amino acid sequence of the CDR-H2 is shown as SEQ ID No. 3 (DPENGD), the amino acid sequence of the CDR-H3 is shown as SEQ ID No. 5 (NPQSERKPSTYSL), the amino acid sequence of the CDR-L1 is shown as SEQ ID No. 7 (KSSQSLLYGDGKTYLN), the amino acid sequence of the CDR-L2 is shown as SEQ ID No. 9 (LVSKLDS), and the amino acid sequence of the CDR-L3 is shown as SEQ ID No. 11 (VQGSYFPLT); the second antibody targeting beta-amyloid 40 comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3), wherein: the amino acid sequence of the CDR-H1 is shown as SEQ ID No. 2 (GPNFKDF), the amino acid sequence of the CDR-H2 is shown as SEQ ID No. 4 (DPENGD), the amino acid sequence of the CDR-H3 is shown as SEQ ID No. 6 (CDAIQRHFIDYDF), the amino acid sequence of the CDR-L1 is shown as SEQ ID No. 8 (KSSQSLLYSDGKTYLN), the amino acid sequence of the CDR-L2 is shown as SEQ ID No. 10 (LVSKLDS), and the amino acid sequence of the CDR-L3 is shown as SEQ ID No. 12 (VQESHFPLT); the first antibody targeting beta-amyloid 42 comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3), wherein: the amino acid sequence of the CDR-H1 is shown as SEQ ID No. 17 (GFNFKDF), the amino acid sequence of the CDR-H2 is shown as SEQ ID No. 19 (DPENGD), the amino acid sequence of the CDR-H3 is shown as SEQ ID No. 21 (HAIWYDEEELYRH), the amino acid sequence of the CDR-L1 is shown as SEQ ID No. 23 (KSSQSLLYSDGKGYLN), the amino acid sequence of the CDR-L2 is shown as SEQ ID No. 25 (LVSKLDS), and the amino acid sequence of the CDR-L3 is shown as SEQ ID No. 27 (VQGSHFPLT); the second antibody targeting beta-amyloid 42 comprises three heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3), wherein: the amino acid sequence of the CDR-H1 is shown as SEQ ID No. 18 (GFNFKDF), the amino acid sequence of the CDR-H2 is shown as SEQ ID No. 20 (EPENGD), the amino acid sequence of the CDR-H3 is shown as SEQ ID No. 22 (SENISYLRRTRGA), the amino acid sequence of the CDR-L1 is shown as SEQ ID No. 24 (KSSQTLLYSDGKTYLN), the amino acid sequence of the CDR-L2 is shown as SEQ ID No. 26 (LVSKLDS), and the amino acid sequence of the CDR-L3 is shown as SEQ ID No. 28 (VQGSHFPLT).

In some embodiments, the first antibody that targets β -amyloid 40 comprises a heavy chain variable region and a light chain variable region, wherein: the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 13 (EVQ LQQSAAELVRTGASVKLSCTTSGFNFKDFYMHWIKQRPEQGLEWIGWLD PENGDTEYAPKFQGKATMAADTSSDTTYLQLSSLMSEDTAVYYCNVNPQ SERKPSTYSLWGQGTTLTVAS), or an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID No. 13, the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 15 (DVMMTGSPHTLTVTIGQPASISCKSSQSLLYGD GKTYLNWLFQRPGQSPKRLIYLVSKLDSGVPDRFTGSGTGTDFTLKISRVE AEDLGIYYCVQGSYFPLTFGAGTKIALKRA), or an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID No. 15; the second antibody targeting beta-amyloid 40 comprises a heavy chain variable region and a light chain variable region, wherein: the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 14 (EVQLQMSAAELVRTGAS VKLSCTTSGPNFKDFYMHWIKQRPEQGLDWIGWLDPENGDTEYAPKFQG KATMAGDTSSDTTYLQLSSLMSEDTAVRYCNVCDAIQRHFIDYDFWGQG TTLTVAS), or an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID No. 14, the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 16 (DVMMTQSPRTISVTIGQPASISCKSSQSLLYSDGKTYLNWLLQRPG QSPKRLIYLVSKLDSGVPDRFTGSGSGTDPTLKISRVEAEDLGIYYCVQES HFPLTFGAGTWLALKRA), or an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID No. 16; the first antibody targeting beta-amyloid 42 comprises a heavy chain variable region and a light chain variable region, wherein: the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 29 (EVQLQQSAGELVRTGASVKLSCATSGFNFK DFYWHWIKQRPEQGLDWIGWLDPENGDTEYAPKFQGKATMAADTSSDT TYLQLSSLMSEETAVYYCNVHAIWYDEEELYRHWGQGTTLTIAS), or an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID No. 29, the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 31 (DVMWT QSPRTLTVTIGQPASISCKSSQSLLYSDGKGYLNWLFQRPGQSPKRLIYLVS KLDSGVPDRFTGSYSGSDFTLKISRVEAEDLGIYYCVQGSHFPLTFGAGTK LAVKRA), or an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID No. 31; the second antibody targeting beta-amyloid 42 comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region has the amino acid sequence shown in SEQ ID No. 30 (EVQLQTSAGELVRTGASIKLSCATSGFNFKDFYWHWLKQRPEQ GLDWIGWLEPENGDTEHAPKFQGKATMAAETSSDTTYLQLSFLMSEETA VYHCNVSENISYLRRTRGAWGQGTTLTIAS), or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID No. 30, or the light chain variable region has the amino acid sequence shown in SEQ ID No. 32 (DVMWTQSPRTLSVTIGQP ASISCKSSQSLLYSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTG SGSNTDFTLKISRVDAEELGIYYCVQGSHFPLTFGAGTKLALKHAD), or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID No. 32.

In some embodiments, the kit of any of the preceding claims for detecting β -amyloid 40 and β -amyloid 42 is used to diagnose alzheimer's disease.

In an eighth aspect, the present application provides the use of a kit for detecting β -amyloid 40 and β -amyloid 42 according to any one of the preceding claims for the preparation of a product for detecting β -amyloid 40, β -amyloid 42 and/or for diagnosing alzheimer's disease.

Examples

The present application will be described with reference to specific examples, but the scope of the present application is not limited thereto. Unless otherwise specified, reagents and equipment used in the following examples are all conventional in the art and are commercially available. The methods used are all routine experimental methods, which can be carried out without any doubt by a person skilled in the art on the basis of the examples and with corresponding results.

EXAMPLE 1 preparation of recombinant antibodies rAbeta 40-A, rA beta 40-B, rA beta 42-A and rAbeta 42-B

The preparation method of the recombinant antibody in the embodiment comprises the following steps: the commercial Abeta 40 antigen (R & D, cat. No. 1191) and the commercial Abeta 42 antigen (R & D, cat. No. 1428) were subjected to oligomerization according to the method described in Kayd R, head E, thompson JL et al Common structure of soluble amyloid oligomers implies common mechanism ofpathogenesis [ J ] Science,2003,300 (5618):486-489. Mice were immunized with either the oligomerized aβ40 or aβ42 antigen, respectively. After obtaining a murine monoclonal antibody cell line with strong affinity for antigen, antibody variable region sequence sequencing was performed. Chimeric vector construction was completed according to variable region sequences and transfected into Hek293T cell lines for recombinant antibody expression. Wherein the light chain vector used was pFDES 2ss-CLIg-mk (constructed using EcoRI and BstAPI cleavage sites) and the heavy chain vector was pFDEs-CHIg-mg 1 (constructed using EcoRI and AfeI cleavage sites), both of which were commercial vectors, and no additional treatment was required. After the recombinant antibodies are expressed in a Hek293T cell line, purified recombinant antibodies rAbeta 40-A, rA beta 40-B, rA beta 42-A and rAbeta 42-B are obtained through Protein G filler. Wherein the amino acid sequence of the rAbeta 40-A heavy chain variable region is shown as SEQ ID No. 13, the amino acid sequence of the rAbeta 40-A light chain variable region is shown as SEQ ID No. 15, the amino acid sequence of the rAbeta 40-B heavy chain variable region is shown as SEQ ID No. 14, the amino acid sequence of the rAbeta 40-B light chain variable region is shown as SEQ ID No. 16, the amino acid sequence of the rAbeta 42-A heavy chain variable region is shown as SEQ ID No. 29, the amino acid sequence of the rAbeta 42-A light chain variable region is shown as SEQ ID No. 31, the amino acid sequence of the rAbeta 42-B heavy chain variable region is shown as SEQ ID No. 30, and the amino acid sequence of the rAbeta 42-B light chain variable region is shown as SEQ ID No. 32.

EXAMPLE 2 verification of the Performance of recombinant antibodies rAbeta 40-A, rA beta 40-B, rA beta 42-A and rAbeta 42-B

Concentration measurements were performed on batches of different recombinant antibodies by means of BCA (Bicinchoninic AcidAssay) technique, the concentration statistics are shown in table 1 below, and the purified recombinant antibody concentrations are shown in table 1. The BCA protein concentration assay kit (enhanced) (bi yun day, cat No. P0010) was used. In particular according to the instruction of the kit.

TABLE 1

Multiple batches of recombinant antibodies were affinity tested by means of the Elisa technique. The ELISA plate was coated with 100ngA beta 40 or Abeta 42 antigen per well, and incubated with the double diluted recombinant antibodies at 37℃for 1 hour. After the incubation, adding goat anti-human secondary antibody marked by HRP, finally adding TMB for color development, adding 2M sulfuric acid for stopping reaction, placing the wavelength of an enzyme label at 450nM, and detecting, wherein the detection results are shown in tables 2-9 respectively.

20220601 batch rAbeta 40-A:

TABLE 2

20220701 batch rAbeta 40-A:

TABLE 3 Table 3

20220601 batch rAbeta 40-B:

TABLE 4 Table 4

20220701 batch rAbeta 40-B:

TABLE 5

20220601 batch rAbeta 42-A:

TABLE 6

20220701 batch rAbeta 42-A:

TABLE 7

20220601 batch rAbeta 42-B:

TABLE 8

20220701 batch rAbeta 42-B:

TABLE 9

The EC50 of each batch of recombinant antibodies was calculated according to SigmaPlot 13.0 software and the data are summarized in table 10 below, the results in table 10 showing that the above recombinant antibodies have higher affinity. Relative to nM K in the prior art _D Value, K of recombinant antibodies of the present application _D The values can reach pM grade, about one thousandth of the prior art, and the affinity of the recombinant antibody is far higher than that of the prior art.

Table 10

Example 3 preparation of kit for detecting beta-amyloid and detection procedure

(1) Preparing a beta-amyloid antibody coated magnetic bead working solution: magnetic beads selected from tosyl activated magnetic beads (Thermo Dynabeads) ^TM M-280Tosylactivated, cat#30110D) at an initial concentration of 100mg/mL. After vortexing, sonication was carried out for 5min and resuspended well. The fully resuspended mother-of-magnetic beads solution was aspirated with a pipette 100. Mu.L (about 10mg of magnetic beads) and the solution was removed by magnetic separation for 5 min. 165. Mu.L of 0.1M borate buffer (pH 9.5) and 200. Mu.g of rAbeta.40-A or rAbeta.42-A recombinant antibodies were added, thoroughly mixed and incubated overnight at 37 ℃. After incubation, magnetically separating for 5minAnd (5) removing liquid. 10% BSA was added and blocked at 37℃for 3h. After closing, the liquid was removed by magnetic separation for 5 min. Finally, buffer TBS-T (0.1M phosphate buffer, pH7.4, supplemented with 0.1% BSA,0.5% Tween-20 and 0.1% Proclin 300) was added and stored at 4deg.C for use. In this example, the mass ratio of the recombinant antibody to the magnetic beads was 1:50. The mother solution after coating is further diluted by 20 times by buffer solution TBS-T to prepare working solution for use.

(2) Preparing beta-amyloid protein labeled antibody working solution: 0.5mg of rAbeta 40-B or rAbeta 42-B recombinant antibody was aspirated and mixed well with 8.25. Mu.L of a 4mM NSP-SA-NHS (Heliosense, cat#HS-11015005 in DMF) solution for 2 hours. 200 μl of 5% DL-lysine was added to the solution and mixing was continued for 30min. Desalting was performed using Sephadex G-25, buffer TBS-T (0.1M phosphate buffer system, pH7.4, supplemented with 0.1% BSA,0.5% Tween-20 and 0.1% Proclin 300). Adding equal volume of glycerol, mixing, and preserving at-20deg.C. The mass ratio of recombinant antibody to NSP-SA-NHS in this example was 1:10. The marked mother solution is further diluted 4000 times by buffer TBS-T to prepare working solution for use.

(3) Tracing a sample: at the position of

G1200 platform is used according to the instruction +.>

Gβ -Amyloid 1-40 (Fujirebio, cat. No. 231524) or +.>

Aβ antigen was traced to Gβ -Amyloid1-42 (Fujirebio, cat. No. 230336).

(4) Split charging of a calibrator and a quality control product: the calibrator and quality control products of Abeta 40 and Abeta 42 are prepared from antigen and sample diluent. Wherein the concentration range of the A beta 40 calibrator is 10-40000pg/mL, and the concentration range of the A beta 42 calibrator is 10-3000pg/mL; the concentration of the A beta 40 quality control product is 50pg/ml,500pg/ml and 20000pg/ml; the concentration of A.beta.42 quality control was 25pg/ml,100pg/ml and 2000pg/ml. The sample dilution was PBS buffer at pH7.4, and the sample dilution also contained 1% BSA and 0.1% proclin-300 per liter.

(5) And (3) assembling a kit: and subpackaging the coated antibodies and the labeled antibodies produced by 3 batches of small-scale tests in a reagent bin, and assembling the coated antibodies and the labeled antibodies, a calibrator and a quality control product into a kit. Specific information is shown in table 11 below:

TABLE 11

(6) On-machine detection

The detection process comprises the following steps: the full-automatic chemiluminescence immunoassay (Smart 500s full-automatic chemiluminescence immunoassay, chongqing Ke Simai biosciences Co., ltd.) is used as a detection tool to check and supplement consumable materials, and the self-detection is completed after the machine is started. The parameters and the reagent lot number required by the Aβ40 and Aβ42 test are input manually or by scanning codes, and the reagent is placed at the corresponding reagent position. The master curves were calibrated using aβ40 and aβ42 calibrators, respectively. And controlling the quality of the detection system by using the quality control products of Abeta 40 and Abeta 42 respectively. And after the quality control is qualified, sequentially adding samples to be detected into the sample positions for detection. And after the detection is finished, carrying out data output, analysis and arrangement.

The reaction system: the machine automatically aspirates 100. Mu.L of sample and mixes it with 100. Mu.L of magnetic bead working solution and 100. Mu.L of labeled antibody working solution thoroughly, and incubates it at 37℃for 15min before magnetic separation. After the supernatant was discarded, the immunoassay kit washing solution (PBS and Tween-20) was added, and the washing was repeated 3 times. The reaction was then sent to a darkroom, and 100. Mu.L each of luminescence excitation solution A (nitric acid and hydrogen peroxide aqueous solution) and luminescence excitation solution B (sodium hydroxide aqueous solution) for the immunoassay instrument was added to perform luminescence reaction, and luminescence values were recorded. And calculating to obtain a corresponding concentration value of the sample by combining the recorded and calibrated calculation curve.

Example 4 optimization of magnetic beads and coated antibody concentration

Tosyl activated beads (Thermo DynabeadsTM M-280Tosylated, cat # 14204) and carboxyl beads (Thermo DynabeadsTM M-270Carboxylic Acid,Cat#14305D) were chosen, vortexed, sonicated for 5min and resuspended thoroughly. Approximately 10mg of the magnetic beads, which were sufficiently resuspended, were each aspirated with a pipette, and the liquid was removed by magnetic separation for 5 min.

Tosyl activated magnetic bead system: 165. Mu.L of 0.1M borate buffer (pH 9.5) and 100. Mu.g, 200. Mu.g or 400. Mu.g of rAbeta.40-A or rAbeta.42-A recombinant antibodies were added and mixed well and incubated overnight at 37 ℃. After incubation, the liquid was removed by magnetic separation for 5 min. 10% BSA was added and blocked at 37℃for 3 hours. After closing, the liquid was removed by magnetic separation for 5 min. Finally, buffer TBS-T (0.1M phosphate buffer, pH7.4, supplemented with 0.1% BSA,0.5% Tween-20 and 0.1% Proclin 300) was added and stored at 4deg.C for use. In this example, the mass ratio of the recombinant antibody to the magnetic beads was 1:50, 1:100, and 1:200, respectively. The coated mother solution is required to be diluted for 20 times continuously to prepare the beta-amyloid antibody coated magnetic bead working solution for use.

Carboxyl magnetic bead system: the beads were resuspended by adding 1mL of 0.1M MES buffer (pH 5.0), 100. Mu.g, 200. Mu.g or 400. Mu.g of rAbeta.40-A or rAbeta.42-A recombinant antibody was added and vortexed. After 30 minutes of spin mixing at room temperature, 100. Mu.L of coupling reagent (10 mg/mL EDC, in ice-cold 0.1M MES buffer pH 5.0) was added and ready to use. And then the mixture is rotated and mixed for 3 hours at room temperature. After incubation, the liquid was removed by magnetic separation for 5 min. Finally, buffer TBS-T (0.1M phosphate buffer, pH7.4, supplemented with 0.1% BSA,0.5% Tween-20 and 0.1% Proclin 300) was added and stored at 4deg.C for use. In this example, the mass ratio of the recombinant antibody to the magnetic beads was 1:50, 1:100, and 1:200, respectively. The coated mother solution is required to be diluted for 20 times continuously to prepare the beta-amyloid antibody coated magnetic bead working solution for use.

The preparation method of the corresponding beta-amyloid protein labeled antibody working solution in the embodiment comprises the following steps: rAbeta 40-B or rAbeta 42-B recombinant antibody (0.25 mg) was aspirated and well mixed with 8.25. Mu.L of a 4mM NSP-SA-NHS (Heliosense, cat#HS-11015005 in DMF) solution for 2 hours. 200 μl of 5% DL-lysine was added to the solution and mixing was continued for 30min. Desalting was performed using Sephadex G-25, buffer TBS-T (0.1M phosphate buffer system, pH7.4, supplemented with 0.1% BSA,0.5% Tween-20 and 0.1% Proclin 300). Adding equal volume of glycerol, mixing, and preserving at-20deg.C. The mass ratio of recombinant antibody to NSP-SA-NHS in this example was 1:20. The marked mother liquid is required to be diluted 4000 times continuously to prepare the beta-amyloid marked antibody working liquid for use.

Antigen tracing was performed with reference to example 3, part (3).

The corresponding machine-on steps in the embodiment are as follows: the machine automatically aspirates 100. Mu.L of sample and mixes it with 100. Mu.L of magnetic bead working solution and 100. Mu.L of labeled antibody working solution thoroughly, and incubates it at 37℃for 25min before magnetic separation. After the supernatant was discarded, the wash solution matched with the immunoassay instrument was added, and the washing was repeated 3 times. Then the reaction was sent to a darkroom, 100. Mu.L each of luminescence excitation liquid A and luminescence excitation liquid B of the immunoassay instrument were added to carry out luminescence reaction, and luminescence values were recorded. And calculating to obtain a corresponding concentration value of the sample by combining the recorded and calibrated calculation curve.

The two different magnetic bead systems and the combination of the concentration of a plurality of different coated antibodies are subjected to cross combination and the linearity of the combination is detected. Each antigen concentration was repeated 3 times per combination and the mean was counted. The results of the measurements are shown in tables 12 and 13 below:

table 12

TABLE 13

The results in tables 12 and 13 above show that tosyl activated bead systems are preferred for rAbeta 40-A and rAbeta 42-A coating processes. The performance is relatively better with an amount of 200 mug or 400 mug of corresponding antibody per 10mg of magnetic beads. It is further preferable to use 200. Mu.g of the recombinant antibody per 10mg of the magnetic beads (mass ratio of the recombinant antibody to the magnetic beads 1:50) in view of the cost of the raw materials.

Example 5 labeled antibody coupling ratio optimization

The coating of antibodies was performed with reference to section (1) of example 3.

rAbeta 40-B or rAbeta 42-B recombinant antibodies (0.1 mg,0.25mg,0.5mg and 0.75 mg) were aspirated, and mixed well with 8.25. Mu.L of a 4mM NSP-SA-NHS (Heliosense, cat#HS-11015005 in DMF) solution for 2 hours, respectively. 200 μl of 5% DL-lysine was added to the solution and mixing was continued for 30min. Desalting was performed using Sephadex G-25, buffer TBS-T (0.1M phosphate buffer system, pH7.4, supplemented with 0.1% BSA,0.5% Tween-20 and 0.1% Proclin 300). Adding equal volume of glycerol, mixing, and preserving at-20deg.C. The mass ratios of recombinant antibody to NSP-SA-NHS in this example were 1:50, 1:20, 1:10, 1:6.7, respectively. The marked mother solution is required to be continuously diluted 4000 times to be prepared into working solution for use.

Antigen tracing was performed with reference to example 3, part (3).

The above-mentioned combination of the labeled antibody coupling ratio and dilution ratio was cross-combined and the linearity thereof was examined. Each antigen concentration was repeated 3 times per combination and the mean was counted. The results of the measurements are shown in tables 14 and 15 below:

TABLE 14

TABLE 15

The results in tables 14 and 15 above show that in rAbeta 40-B and rAbeta 42-B labeling processes, the kit detection sensitivity can be further improved when the amount of labeled antibody used is increased to 0.5mg or 0.75 mg. It is further preferable that the amount of the labeled antibody used is 0.5mg, i.e., the ratio of the amount of the labeled antibody (3.3 nmol) to the amount of the NSP-SA-NHS (33 nmol) is 1:10, in view of the cost of the raw materials.

Example 6 run-on detection procedure optimization

Coating of antibodies was performed as described in example 3, labeling of antibodies was performed as described in example 3, section (2), and antigen tracing was performed as described in example 3, section (3).

The corresponding machine-on steps in the embodiment are as follows: the machine automatically aspirates 50. Mu.L, 100. Mu.L or 150. Mu.L of cerebrospinal fluid samples and mixes them well with 100. Mu.L of magnetic bead working fluid and 100. Mu.L of labeled antibody working fluid, and incubates them at 37℃for 10min, 15min, 25min and 40min before performing magnetic separation. After the supernatant was discarded, the wash solution matched with the immunoassay instrument was added, and the washing was repeated 3 times. Then the reaction was sent to a darkroom, 100. Mu.L each of luminescence excitation liquid A and luminescence excitation liquid B of the immunoassay instrument were added to carry out luminescence reaction, and luminescence values were recorded. And (3) calculating to obtain a corresponding concentration value (pg/mL) of the sample by combining the recorded and calibrated calculation curve.

10 cases of the different upper programs are respectively carried out

G1200 platform traceable cerebrospinal fluid sample detection, analysis of the deviation of the detection result and assigned value. The results of the measurements are shown in tables 16 to 21 below:

table 16

TABLE 17

TABLE 18

The results in tables 16 to 18 show that the A.beta.40 upper machine program combination meeting R2 > 0.90 is 100. Mu.L or 150. Mu.L in sample size, and the reaction time is 15min, 25min or 40min. A final loading procedure was performed with an A.beta.40 sample size of at least 100. Mu.L and a reaction time of at least 15 min.

TABLE 19

Table 20

Table 21

The results in tables 19 to 21 show that the A.beta.42 upper machine program combination satisfying R2 > 0.90 is 100. Mu.L or 150. Mu.L in sample size, and the reaction time is 15min, 25min or 40min. A preferred procedure is to load A.beta.42 in an amount of at least 100. Mu.L and a reaction time of at least 15 min.

Example 7 evaluation of kit Performance

(1) Accuracy of

The traced Abeta 40 and Abeta 42 antigens were respectively prepared into 10000pg/mL and 800pg/mL solutions, and the detection was repeated 3 times and data analysis was performed. The results of the detection accuracy of the Abeta 40 kit and the Abeta 42 kit are shown in tables 22 and 23, respectively (concentration unit is pg/mL).

Table 22

Kit lot	Detection result 1	Detection result 2	Detection result 3	Mean value of	Marking value	Deviation of
							20220901	10430.91	9881.134	10376.05	10229.36	10000	2.29％
20220902	10274.38	10229.46	10140.4	10214.74	10000	2.15％
							20220903	9720.783	9608.919	9573.719	9634.474	10000	-3.66％

Table 23

Kit lot	Detection result 1	Detection result 2	Detection result 3	Mean value of	Marking value	Deviation of
							20220901	853.468	820.23	834.471	836.0563	800	4.51％
20220902	794.085	865.992	843.758	834.6117	800	4.33％
							20220903	821.309	824.74	837.028	827.6923	800	3.46％

In summary, the relative deviation of the measurement results is within a range of +/-10%, and 3 results meet the requirements, and the accuracy of the above 3 batches of kits is qualified.

(2) Precision of

According to the protocol of the American clinical laboratory standards Committee (NCCLS/CLSI) document EP5-A2, the in-batch precision and inter-laboratory precision are tested by adopting a multi-factor integrated nested design, different operators, different equipment and different places are tested once each day in the morning and afternoon, each sample is tested repeatedly for 2 times, the test is continuously carried out for 20 days, 160 data results are collected for each batch of the kit, and the precision is calculated. Meanwhile, the Aβ40 antigen after tracing is configured into 500pg/mL and 30000pg/mL solutions, and the Aβ42 antigen after tracing is configured into 100pg/mL and 2000pg/mL solutions. The test was repeated 3 times and data analysis was performed. The results of the measurements are shown in tables 24 and 25 below:

Table 24

Table 25

In conclusion, the detection results of the two kits repeatedly carried out on the low-concentration sample and the high-concentration sample show that the precision CV in the batch is less than 5%, and the precision CV between batches is less than 10%, which indicates that the two kits meet the precision performance evaluation requirement.

(3) Linear range

1 cerebrospinal fluid supplemented with Abeta 40 or Abeta 42 antigen was selected, respectively, diluted with a diluent at 12 concentration levels, and each diluted sample was tested repeatedly 3 times to calculate the mean value. The corresponding linear relationship was calculated from the concentration points at which the results gradually decreased, and the widest linear range of the kit was determined, and the detection results of the aβ40 kit and the aβ42 kit are shown in tables 26 and 27, respectively (concentration units are pg/mL).

Table 26

Table 27

Tables 26 and 27 above show that the A.beta.40 kit has a correlation coefficient r of not less than 0.9900 and no outlier in the range of [10 to 40000] pg/mL. The correlation coefficient r of the Abeta 42 kit is not lower than 0.9900 within the range of [ 10-3000 ] pg/mL and has no outlier. Therefore, the linear range of the A beta 40 kit is [ 10-40000 ] pg/mL, and the linear range of the A beta 42 kit is [ 10-3000 ] pg/mL.

(4) Sensitivity of

And (5) detecting the blank sample by using the kit, and repeating the detection for 20 times to determine the sensitivity. The results of the Abeta 40 kit and Abeta 42 kit detection are shown in tables 28 and 29 (concentration units are pg/mL) below, respectively:

Table 28

Project	Parameters (parameters)
		Mean	741.5
SD	23.668
		Mean+2SD	788.835
Corresponding concentration value	4.71pg/mL

The average value of the average luminescence value of the blank sample measured by the Abeta 40 kit is 741.5, and the average value is brought into a reaction curve, so that the sensitivity of the Abeta 40 kit is 4.71pg/mL.

Table 29

Project	Parameters (parameters)
		Mean	346
SD	11.399
		Mean+2SD	368.798
Corresponding concentration value	4.38pg/mL

The average luminescence value of the blank sample measured by the Abeta 42 kit is 346, and the average luminescence value is brought into a reaction curve, so that the sensitivity of the Abeta 42 kit is 4.38pg/mL.

(5) Sample detection results

Detecting warp

Samples 40 of the G1200 platform assignment were used to determine the consistency of the kit with the platform product. And detecting the part beyond the linear range of the target reagent by adopting a dilution and loading mode. The results of the aβ40 consistency analysis are shown in fig. 1 (the concentration unit is pg/mL), and the regression analysis data show that y=0.9847x+493.46 and r2= 0.9289, which proves that the product has good consistency with commercial products. The results of the aβ42 consistency analysis are shown in fig. 2 (the concentration unit is pg/mL), and the regression analysis data show that y=0.9162x+61.845 and r2= 0.9227, which proves that the product has good consistency with commercial products.

According to the embodiments, the kit has high accuracy, high precision and high sensitivity, the linear range of the A beta 40 kit is [ 10-40000 ] pg/mL, the linear range of the A beta 42 kit is [ 10-3000 ] pg/mL, the linear range of the kit is far wider than the linear range of the prior art, the detection upper limit of the kit is more than twice the detection upper limit of the prior art, and meanwhile, the kit and a commercial product of the kit have good consistency.

The above description is only of the preferred embodiments of the present application and is not intended to limit the present application in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to the equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present application still fall within the protection scope of the technical solution of the present application.

Claims

1. A kit for detecting β -amyloid 40, comprising:

magnetic beads coated with a first antibody targeting beta-amyloid 40; and

a chemiluminescent-labeled second antibody targeting beta-amyloid 40;

2. The kit according to claim 1, wherein,

the amino acid sequence of the CDR-H1 is shown as SEQ ID No. 1;

The amino acid sequence of the CDR-H2 is shown as SEQ ID No. 3;

the amino acid sequence of the CDR-H3 is shown as SEQ ID No. 5;

the amino acid sequence of the CDR-L1 is shown as SEQ ID No. 7,

the amino acid sequence of the CDR-L2 is shown as SEQ ID No. 9;

the amino acid sequence of the CDR-L3 is shown as SEQ ID No. 11;

the amino acid sequence of the CDR-H1 is shown as SEQ ID No. 2;

the amino acid sequence of the CDR-H2 is shown as SEQ ID No. 4;

the amino acid sequence of the CDR-H3 is shown as SEQ ID No. 6;

the amino acid sequence of the CDR-L1 is shown as SEQ ID No. 8;

the amino acid sequence of the CDR-L2 is shown as SEQ ID No. 10;

the amino acid sequence of the CDR-L3 is shown as SEQ ID No. 12.

3. Use of a kit according to claim 1 or 2 for the preparation of a product for the detection of beta-amyloid 40 and/or for the diagnosis of alzheimer's disease.

4. A method for detecting the amount of beta-amyloid 40 in a sample comprising the steps of:

5. A kit for detecting β -amyloid 42, comprising:

magnetic beads coated with a first antibody targeting beta-amyloid 42; and

a chemiluminescent-labeled second antibody targeting beta-amyloid 42;

6. The kit according to claim 5, wherein,

the amino acid sequence of the CDR-H1 is shown as SEQ ID No. 17;

the amino acid sequence of the CDR-H2 is shown as SEQ ID No. 19;

the amino acid sequence of the CDR-H3 is shown as SEQ ID No. 21;

the amino acid sequence of the CDR-L1 is shown as SEQ ID No. 23,

The amino acid sequence of the CDR-L2 is shown as SEQ ID No. 25;

the amino acid sequence of the CDR-L3 is shown as SEQ ID No. 27;

the amino acid sequence of the CDR-H1 is shown as SEQ ID No. 18;

the amino acid sequence of the CDR-H2 is shown as SEQ ID No. 20;

the amino acid sequence of the CDR-H3 is shown as SEQ ID No. 22;

the amino acid sequence of the CDR-L1 is shown as SEQ ID No. 24;

the amino acid sequence of the CDR-L2 is shown as SEQ ID No. 26;

the amino acid sequence of the CDR-L3 is shown as SEQ ID No. 28.

7. Use of a kit according to claim 5 or 6 for the preparation of a product for the detection of beta-amyloid 42 and/or for the diagnosis of alzheimer's disease.

8. A method for detecting the amount of beta-amyloid 42 in a sample comprising the steps of:

9. A kit for detecting β -amyloid 40 and β -amyloid 42, comprising:

magnetic beads coated with a first antibody targeting beta-amyloid 40;

a chemiluminescent-labeled second antibody targeting beta-amyloid 40;

magnetic beads coated with a first antibody targeting beta-amyloid 42; and

a chemiluminescent-labeled second antibody targeting beta-amyloid 42;

10. Use of the kit of claim 9 for the preparation of a product for detecting beta-amyloid 40, beta-amyloid 42 and/or for diagnosing alzheimer's disease.